Process for the manufacture of D-pantothene acid and/or its salts by fermentation

ABSTRACT

The invention provides a process for the preparation of D-pantothenic acid and/or salts thereof or feedstuffs additives comprising these by fermentation of microorganisms of the Enterobacteriaceae family, in particular those which already produce D-pantothenic acid, characterized in that the nucleotide sequence(s) in the microorganisms which code(s) for the pckA gene is (are) attenuated, in particular eliminated.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to DE 101 12 100.8, filed Mar.14, 2001 and to U.S. provisional application 60/304,774, filed Jul. 13,2001. The entire contents of both documents are incorporated byreference.

REFERENCE TO SEQUENCE LISTING

[0002] The contents of the Sequence Listing in computer readable form asprovided herewith are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] An improved process for the fermentive preparation ofD-pantothenic acid and its derivatives using a microorganism in whichthe pckA gene is attenuated or eliminated, especially microorganisms ofthe Enterobacteriaceae family. Nucleic acids and vectors encoding anattenuated pckA gene, and host cells comprising such nucleic acids orvectors, or in which the pckA gene has been eliminated.

[0005] 2. Description of Related Art

[0006] World-wide production of pantothenic acid is the order ofmagnitude of several thousand tons a year. Pantothenic acid has avariety of uses, including in medical, pharmaceutical and nutritionalproducts, including foodstuffs or feedstuffs. Additionally, asignificant portion of the pantothenic acid produced is used for thenutrition of stock animals such as poultry and pigs.

[0007] While pantothenic acid can be prepared by chemical synthesis,chemical synthesis results in the production of a racemic mixture ofDL-pantothenic acid, which must be further purified to obtain thenaturally occurring, more biologically active D-pantothenic acid.DL-pantolactone is an important precursor of DL-pantothenic acid in thechemical synthesis of DL-pantothenic acid and may be prepared in amulti-stage process from formaldehyde, isobutylaldehyde and cyanide. Theresulting racemic mixture of DL-pantolactone is subsequently separatedand subjected to a condensation reaction with β-alanine. D-pantothenicacid is subsequently obtained.

[0008] Pantothenic acid may also be prepared by fermentation using asuitable microorganism. One advantage of the fermentative preparation bymicroorganisms lies in the direct formation of the desiredstereoisomeric form, that is to say the D-form, which is free fromL-pantothenic acid.

[0009] D-pantothenic acid can be produced by various types of bacteria,such as Escherichia coli (E. coli), Arthrobacter ureafaciens,Corynebacterium erythrogenes, Brevibacterium ammoniagenes, and also byyeasts, such as Debaromyces castellii. Typical nutrient media orsolutions comprise glucose, DL-pantoic acid and β-alanine, as shown inEP-A 0 493 060. EP-A 0 493 060 also shows that in E. coli the formationof D-pantothenic acid is improved by the amplification of E. colipantothenic acid biosynthesis genes contained on plasmids pFV3 and pFV5,and use of a nutrient solution comprising glucose, DL-pantoic acid andβ-alanine.

[0010] EP-A 0 590 857 and U.S. Pat. No. 5,518,906 describe mutantsderived from E. coli strain IF03547, such as FV5714, FV525, FV814,FV521, FV221, FV6OSl and FV5069, which carry resistances to variousantimetabolites, such as salicylic acid, α-ketobutyric acid,β-hydroxyaspartic acid, O-methylthreonine and α-ketoisovaleric acid.Such strains or mutants produce pantoic acid in a nutrient solutioncomprising glucose, and produce D-pantothenic acid in a nutrientsolution comprising glucose and β-alanine.

[0011] EP-A 0 590 857 and U.S. Pat. No. 5,518,906 indicate that afteramplification of the pantothenic acid biosynthesis genes panB, panC andpanD, which are said to be contained on the plasmid pFV31, in theabove-mentioned strains the production of D-pantoic acid in nutrientsolutions comprising glucose and the production of D-pantothenic acid ina nutrient solution comprising glucose and β-alanine is improved.

[0012] The favorable effect of enhancement of the ilvGM operon onproduction of D-pantothenic acid is also reported in WO97/10340.Finally, the effect of enhancement of the panE gene on the formation ofD-pantothenic acid is reported in EP-A-1001027.

[0013] D-pantothenic acid or the corresponding salt may be isolated fromthe fermentation broth and purified (EP-A-0590857 and WO96/33283) andsubsequently used in purified form, or alternatively, the fermentationbroth comprising D-pantothenic acid may be dried (EP-A-1050219) and usedin particular as a feedstuffs additive.

[0014] In view of the importance of D-pantothenic acid, its would behighly desirable to have a more simple, economical and efficient processthat produces high yields of D-pantothenic acid or its derivatives.

BRIEF SUMMARY OF THE INVENTION

[0015] The present invention provides a more simple, improved,economical and efficient process for producing D-pantothenic acid byfermentive production using a microorganism selected or modified tocontain an attenuated pckA gene or a microorganism in which the pckAgene has been eliminated.

[0016] Another object of the present invention is a process thatprovides high yields of D-pantothenic acid useful in pharmaceuticalproducts, foods, nutritional products or animal feeds.

[0017] An additional object of the invention is to provide a derivativeof pantothenic acid, such as a calcium salt, that is more stable oreasily handled or processed that D-pantothenic acid (free acid).

[0018] Yet another object of the invention is provision of improvedanimal feedstocks or feedstock additives containing high amounts ofD-pantothenic acid, as well as feedstocks or feedstock additives inwhich the content of D-pantothenic acid or its derivative is more stableor biologically available. Thus, for example, the invention provides aprocess in which, after conclusion of the fermentation, all or some ofthe biomass remains in the fermentation broth, and the broth obtained inthis way is processed, optionally after concentration, to a solidmixture which comprises D-pantothenic acid and/or salts thereof and alsocomprises further constituents of the fermentation broth.

[0019] The invention also includes the formulation or supplementation ofproducts, such as pharmaceuticals, nutritional products, cosmetics,foods, or feedstuffs using either the isolated and purifiedD-pantothenic acid or one or more of its derivatives, or alternatively,the biomass or dried and/or concentrated fermentation broth or culturemedium.

[0020] Other objects of the invention include nucleic acids and vectorsthat encode attenuated pckA genes, as well as cells, such as E. coli,containing such genes or vectors.

BRIEF DESCRIPTION OF THE DRAWINGS (FIGURES)

[0021]FIG. 1: pMAK705ΔpckA (=pMAK705deltapckA)

[0022]FIG. 2: pTrc99AilvGM

[0023]FIG. 3: pFV31ilvGM

[0024] The length data are to be understood as approx. data. Theabbreviations and designations used have the following meaning:

[0025] cat: Chloramphenicol resistance gene

[0026] rep-ts: Temperature-sensitive replication region of the plasmidpSC101

[0027] pck1: Part of the 5′ region of the pckA gene

[0028] pck2: Part of the 3′ region of the pckA gene

[0029] Amp: Ampicillin resistance gene

[0030] lacI: Gene for the repressor protein of the trc promoter

[0031] Ptrc: trc promoter region, IPTG-inducible

[0032] ilvG: Coding region of the large subunit of acetohydroxy acidsynthase II

[0033] ilvM: Coding region of the small subunit of acetohydroxy acidsynthase II

[0034] 5S: 5S rRNA region

[0035] rrnBT: rRNA terminator region

[0036] panB: Coding region of the panB gene

[0037] panC: Coding region of the panC gene

[0038] The abbreviations for the restriction enzymes have the followingmeaning

[0039] BamHI: Restriction endonuclease from Bacillus amyloliquefaciens

[0040] BglII: Restriction endonuclease from Bacillus globigii

[0041] ClaI: Restriction endonuclease from Caryphanon latum

[0042] EcoRI: Restriction endonuclease from Escherichia coli

[0043] EcoRV: Restriction endonuclease from Escherichia coli

[0044] HindIII: Restriction endonuclease from Haemophilus influenzae

[0045] KpnI: Restriction endonuclease from Klebsiella pneumoniae

[0046] PstI: Restriction endonuclease from Providencia stuartii

[0047] PvuI: Restriction endonuclease from Proteus vulgaris

[0048] SacI: Restriction endonuclease from Streptomyces achromogenes

[0049] SalI: Restriction endonuclease from Streptomyces albus

[0050] SmaI: Restriction endonuclease from Serratia marcescens

[0051] SphI: Restriction endonuclease from Streptomyces phaeochromogenes

[0052] SspI: Restriction endonuclease from Sphaerotilus species

[0053] XbaI: Restriction endonuclease from Xanthomonas badrii

[0054] XhoI: Restriction endonuclease from Xanthomonas holcicola

DETAILED DESCRIPTION OF THE INVENTION

[0055] The terms “D-pantothenic acid”, “pantothenic acid” or“pantothenate” as used in this disclosure encompass both the free acidsand salts of D-pantothenic acid, such as the calcium, sodium, ammoniumor potassium salts. Pantothenic acid derivatives, such as the alcohol,aldehyde, alcohol esters or acid esters, which may be bioconverted ormetabolized into pantothenic acid once ingested or administered to aliving organism, such as a mammal, are also contemplated.

[0056] The term “pckA gene” is known in the art. This gene codes forphosphoenol pyruvate carboxylase (EC 4.1.1.49). An exemplary pckA geneis that of Escherichia coli. The nucleotide sequence of the pckA gene ofEscherichia coli has been published by Medina et al., Journal ofBacteriology 172, 7151-7156 (1990) and can also be found in the genomesequence of Escherichia coli published by Blattner et al., Science 277,1453 - 1462 (1997) under Accession Number AE000416. This term is alsointended to include various allelic forms of this gene as well asmodified versions of this gene as described below.

[0057] The term “attenuated microorganism” refers to one in which theamount or activity of one or more enzymes or other biologically activeproteins is reduced or eliminated. For instance, a microorganism with anattenuated pckA gene may either be deleted for the pckA gene, carry amodified form of the pckA gene that encodes an enzyme with loweredactivity, or may carry a pckA gene with regulatory sequences thatdecrease its expression. The term “attenuation” in this connectiondescribes the reduction or elimination of the intracellular activity ofone or more enzymes (proteins) in a microorganism which are coded by thecorresponding DNA, for example, by using a weak promoter or using a geneor allele which codes for a corresponding enzyme (protein) with a lowactivity or inactivates the corresponding gene or enzyme (protein), oroptionally combining these measures.

[0058] By attenuation measures, including reduction or elimination ofexpression, the activity or concentration of the corresponding proteinis reduced by at least 0-75%, 0-50%, 0 to 25%, 0 to 10%, 0 to 5%, or 0to 1% of the activity or concentration of the wild-type protein or ofthe activity or concentration of the protein in the startingmicroorganism.

[0059] The improved process of the present invention producesD-pantothenic acid or it salts or derivatives using a microorganism inwhich the pckA gene has been attenuated or deleted. Advantageously, amicroorganism of the Enterobacteriaceae family is used, for instance, anE. coli strain. Advantageously, a microorganism already known to produceD-pantothenic acid can be modified to attenuate or eliminate the pckAgene.

[0060] Advantageously, the inventive process may be characterized by thefollowing:

[0061] a) Fermentation of a microorganism of the Enterobacteriaceaefamily in which the pckA gene is attenuated (or eliminated) in a culturemedium suitable for the production of D-pantothenic acid. Optionally,the microorganism used may contain other attenuated or enhanced genesthat improve the yield, stability or efficiency of production ofD-pantothenic acid or its derivatives.

[0062] b) Fermentation may optionally be conducted in the presence ofone or more alkaline earth metal compound(s), which may be addedcontinuously or discontinuously to the culture medium preferably in astoichiometric amount.

[0063] c) Concentration of the D-pantothenic acid or its correspondingsalt or other derivative produced by the fermentation process may beaccomplished by further processing of the culture medium, fermentationbroth or microorganisms used in the fermentation process.

[0064] d) Upon conclusion of the fermentation, the D-pantothenic acid,or its corresponding salt or derivative, may be further purified orisolated.

[0065] Optionally, further modification or derivativization, such asesterification of the panthothenic acid or its salt may be conducted,for instance, to improve stability, handling properties or absorption ofthis compound. Such modifications may be made either as part of thefermenation process or after concentration, isolation or purification ofD-pantothenic acid or its salt. For instance, D-pantothenic acid may befurther converted into a more stable derivative, such as the alcoholderivative pantothenol.

[0066] The microorganisms which the present invention provides canproduce D-pantothenic acid from a variety of carbohydrates, sugars orother carbon-containing substrates, including glucose, sucrose, lactose,fructose, maltose, molasses, starch, cellulose or from glycerol andethanol.

[0067] These strains are representatives of Enterobacteriaceae, inparticular of the genus Escherichia. Advantageously, from the genusEscherichia, the species Escherichia coli is to be mentioned inparticular. Within the species Escherichia coli there may be mentionedthe so-called K-12 strains, such as e.g. the strains MG1655 or W3110(Neidhard et al.: Escherichia coli and Salmonella. Cellular andMolecular Biology (ASM Press, Washington D.C.)) or the Escherichia coliwild type strain IFO3547 (Institute of Fermentation, Osaka, Japan) andmutants derived from these, which have the ability to produceD-pantothenic acid.

[0068] Suitable D-pantothenic acid-producing strains of the genusEscherichia, in particular of the species Escherichia coli, are, forexample

[0069]Escherichia coli FV5069/pFV31

[0070]Escherichia coli FV5069/pFV202

[0071]Escherichia coli FE6/pFE80 and

[0072]Escherichia coli KE3

[0073] It has been found that Enterobacteriaceae produce D-pantothenicacid in an improved manner after attenuation of the pckA gene, whichcodes for phosphoenol pyruvate carboxykinase (EC 4.1.1.49). However, thepresent invention may use any suitable pckA gene, including thosedescribed in the text references mentioned above. Alleles of the pckAgene, which result from the degeneracy of the genetic code or due tosense mutations of neutral function, can also be used. Moreover, nucleicacid sequences which cross-hybridize to the pckA genes described aboveunder stringent conditions and that encode polypeptides havingphospholenol pyruvate activity may also be used, for instance, a nucleicacid sequence that (a) encodes a pckA gene product with reducedenzymatic activity compared to the gene product encoded by SEQ ID NO: 1and (b) that is at least 70%, 80%, 90%, 95% or 99% similar to that ofSEQ ID NO: 1 or that hybridizes with SEQ ID NO: 1 under stringentconditions, wherein stringent conditions comprise washing in 5×SSC at atemperature ranging from 50° to 68° C.

[0074] Homology, sequence similarity or sequence identity of nucleotideor amino acid sequences may be determined conventionally by using knownsoftware or computer programs such as the BestFit or Gap pairwisecomparison programs (GCG Wisconsin Package, Genetics Computer Group, 575Science Drive, Madison, Wis. 53711). BestFit uses the local homologyalgorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of identity or similaritybetween two sequences. Gap performs global alignments: all of onesequence with all of another similar sequence using the method ofNeedleman and Wunsch, J. Mol. Biol. 48:443-453 (1970). When using asequence alignment program such as BestFit, to determine the degree ofsequence homology, similarity or identity, the default setting may beused, or an appropriate scoring matrix may be selected to optimizeidentity, similarity or homology scores. Similarly, when using a programsuch as BestFit to determine sequence identity, similarity or homologybetween two different amino acid sequences, the default settings may beused, or an appropriate scoring matrix, such as blosum45 or blosum80,may be selected to optimize identity, similarity or homology scores.

[0075] Attenuation may be achieved by reducing the expression of thepckA gene or by reducing or eliminating the catalytic properties of theenzyme it encodes. These measures may also be combined.

[0076] The reduction in gene expression can take place by suitableculturing, by genetic modification (mutation) of the signal structuresof gene expression or also by the antisense-RNA technique. Signalstructures of gene expression are, for example, repressor genes,activator genes, operators, promoters, attenuators, ribosome bindingsites, the start codon and terminators. Methods for reducing orenhancing gene expression are known in the art, however, pertinentinformation may be also found, inter alia, in Jensen and Hammer,Biotechnology and Bioengineering 58: 191-195 (1998), in Carrier andKeasling, Biotechnology Progress 15, 58-64 (1999), Franch and Gerdes,Current Opinion in Microbiology 3, 159-164 (2000) and in known textbooksof genetics and molecular biology, such as, for example, the textbook ofKnippers, “Molekulare Genetik [Molecular Genetics]”, 6th edition, GeorgThieme Verlag, Stuttgart, Germany, (1995) or that of Winnacker, “Geneund Klone [Genes and Clones]”, VCH Verlagsgesellschaft, Weinheim,Germany, (1990).

[0077] Certain mutations, which lead to a change or reduction in thecatalytic properties of enzyme proteins, are known from the prior art.Examples which may be mentioned are the works of Qiu and Goodman,Journal of Biological Chemistry 272: 8611-8617 (1997), Yano et al.,Proceedings of the National Academy of Sciences, USA 95, 5511-5515(1998), Wente and Schachmann, Journal of Biological Chemistry 266,20833-20839 (1991). Summarizing descriptions can be found in knowntextbooks of genetics and molecular biology, such as e.g. that byHagemann, “Allgemeine Genetik [General Genetics]”, Gustav FischerVerlag, Stuttgart, (1986).

[0078] Possible mutations include transitions, transversions, insertionsand deletions. Depending on the effect of the amino acid exchange on theenzyme activity, “missense mutations” or “nonsense mutations” arereferred to. Insertions or deletions of at least one base pair in a genelead to “frame shift mutations”, which lead to incorrect amino acidsbeing incorporated or translation being interrupted prematurely.Deletions of several codons typically lead to a complete loss of theenzyme activity. Instructions on generation of such mutations are priorart and can be found in known textbooks of genetics and molecularbiology, such as e.g. the textbook by Knippers, “Molekulare Genetik[Molecular Genetics]”, 6th edition, Georg Thieme Verlag, Stuttgart,Germany, (1995), that by Winnacker, “Gene und Klone [Genes and Clones]”,VCH Verlagsgesellschaft, Weinheim, Germany, (1990) or that by Hagemann,“Allgemeine Genetik [General Genetics]”, Gustav Fischer Verlag,Stuttgart, (1986).

[0079] Suitable mutations in the pckA gene, such as, for example,deletion mutations, can be incorporated into suitable strains by gene orallele replacement.

[0080] A conventional method is the method, described by Hamilton etal., Journal of Bacteriology 174, 4617-4622 (1989), of gene replacementwith the aid of a conditionally replicating pSC101 derivative pMAK705.Other methods described in the prior art, such as, for example, those ofMartinez-Morales et al., Journal of Bacteriology 1999: 7143-7148 (1999)or those of Boyd et al., Journal of Bacteriology 182, 842-847 (2000),can likewise be used.

[0081] It is also possible to transfer mutations in the pckA gene ormutations that affect expression of the pckA gene into various strainsby conjugation or transduction.

[0082] In addition to the attenuation of the pckA gene to increase theamount, simplicity or efficiency of the production of D-pantothenicacid, other enhanced genes may be added to a microbial strain, such as astrain of the Enterobacteriaceae family. For instance, one or more ofthe following genes may be enhanced or over-expressed in an appropriatemicrobial strain:

[0083] the ilvGM operon which codes for acetohydroxy-acid synthase II(WO 97/10340)

[0084] the panB gene which codes for ketopantoate hydroxymethyltransferase (U.S. Pat. No. 5,518,906),

[0085] the panE gene which codes for ketopantoate reductase(EP-A-1001027)

[0086] the panD gene which codes for aspartate decarboxylase (U.S. Pat.No. 5,518,906)

[0087] the panC gene which codes for pantothenate synthetase (U.S. Pat.No. 5,518,906)

[0088] the serC gene which codes for phosphoserine transaminase (Duncanand Coggins, Biochemical Journal 234:49-57 (1986)),

[0089] the gcvT, gcvH and gcvP genes which code for the glycine cleavagesystem (Okamura-Ikeda et al., European Journal of Biochemistry 216,539-548 (1993)), and

[0090] the glyA gene which codes for serine hydroxymethyl transferase(Plamann et al., Nucleic Acids Research 11(7):2065-2075(1983)).

[0091] The term “enhancement” in this connection describes the increasein the intracellular activity of one or more enzymes or proteins in amicroorganism which are encoded by the corresponding DNA. Suchenhancement may be achieved, for example, by increasing the number ofcopies of the gene or genes, using a potent promoter or a gene or allelewhich codes for a corresponding enzyme or protein with a high activity,or optionally combining these measures.

[0092] By enhancement measures, in particular over-expression, theactivity or concentration of the corresponding protein is in generalincreased by at least 1%, 5%, 10%, 25%, 50%, 75%, 100%, 150%, 200%,300%, 400% or 500%, including 1000% or 2000%, based on that of thewild-type protein or the activity or concentration of the protein in thestarting microorganism.

[0093] Finally, it may be advantageous for the production ofD-pantothenic acid with microbial stains, such as strains of theEnterobacteriaceae family, in addition to the attenuation of the pckAgene, for particular genes to be attenuated, eliminated or expressed ata low level. Such genes include:

[0094] the avtA gene which codes for transaminase C (EP-A-1001027) and

[0095] the poxB gene which codes for pyruvate oxidase (Grabau andCronan, Nucleic Acids Research. 14 (13), 5449-5460 (1986)).

[0096] In addition to the attenuation of the pckA gene it mayfurthermore be advantageous for the production of D-pantothenic acid toeliminate undesirable side reactions (Nakayama: “Breeding of Amino AcidProducing Microorganisms”, in: Overproduction of Microbial Products,Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).Microbes, such as bacteria in which the metabolic pathways that reducethe formation of D-pantothenic acid are at least partly eliminated, canbe employed in the process according to the invention.

[0097] Any suitable cultivation method may be selected. For instance,the microorganisms produced according to the invention can be culturedin the batch process (batch culture), the fed batch (feed process) orthe repeated fed batch process (repetitive feed process). A summary ofknown culture methods is described in the textbook by Chmiel(Bioprozesstechnik 1. Einführung in die Bioverfahrenstechnik [BioprocessTechnology 1. Introduction to Bioprocess Technology, Gustav FischerVerlag, Stuttgart, (1991) or in the textbook by Storhas, Bioreaktorenund periphere Einrichtungen [Bioreactors and Peripheral Equipment](Vieweg Verlag, Braunschweig/Wiesbaden (1994).

[0098] The culture medium to be used must meet the requirements of theparticular strains in a suitable manner. Descriptions of culture mediafor various microorganisms are contained in the handbook “Manual ofMethods for General Bacteriology” of the American Society forBacteriology (Washington D.C., USA (1981). Sugars and carbohydrates,such as e.g. glucose, sucrose, lactose, fructose, maltose, molasses,starch and cellulose, oils and fats, such as soya oil, sunflower oil,groundnut oil and coconut fat, fatty acids, such as palmitic acid,stearic acid and linoleic acid, alcohols, such as glycerol and ethanol,and organic acids, such as acetic acid, can be used as the source ofcarbon. These substances can be used individually or as a mixture.

[0099] Organic nitrogen-containing compounds, such as peptones, yeastextract, meat extract, malt extract, corn steep liquor, soya bean flourand urea, or inorganic compounds, such as ammonium sulfate, ammoniumchloride, ammonium phosphate, ammonium carbonate and ammonium nitrate,can be used as the source of nitrogen. The sources of nitrogen can beused individually or as a mixture.

[0100] Phosphoric acid, potassium dihydrogen phosphate or dipotassiumhydrogen phosphate or the corresponding sodium-containing salts can beused as the source of phosphorus.

[0101] The culture medium must furthermore comprise salts of metals,such as magnesium sulfate or iron sulfate, which are necessary forgrowth. Finally, essential growth substances, such as amino acids andvitamins, can be employed in addition to the above-mentioned substances.Precursors of pantothenic acid, such as aspartate, β-alanine,ketoisovalerate, ketopantoic acid or pantoic acid and optionally saltsthereof, can moreover be added to the culture medium. The startingsubstances mentioned can be added to the culture in the form of a singlebatch, or can be fed in during the culture in a suitable manner.

[0102] Basic compounds, such as sodium hydroxide, potassium hydroxide,ammonia or aqueous ammonia, or acid compounds, such as phosphoric acidor sulfuric acid, can be employed in a suitable manner to control the pHof the culture.

[0103] For the preparation of an alkaline earth metal salt ofpantothenic acid, in particular the calcium salt, it is equally possibleto add the suspension or solution of an inorganic compound containing analkaline earth metal, such as, for example, calcium hydroxide, or of anorganic compound, such as the alkaline earth metal salt of an organicacid, for example calcium acetate, continuously or discontinuouslyduring the fermentation. In this manner, the cation necessary forpreparation of the desired alkaline earth metal salt of D-pantothenicacid is introduced into the fermentation broth directly in the desiredamount, preferably in stoichiometric amounts.

[0104] Antifoams, such as fatty acid polyglycol esters, can be employedto control the development of foam. Suitable substances having aselective action, e.g. antibiotics, can be added to the medium tomaintain the stability of plasmids. To maintain aerobic conditions,oxygen or oxygen-containing gas mixtures, such as e.g. air, areintroduced into the culture. The temperature of the culture is usually25° C. to 45° C., and preferably 30° C. to 40° C. Culturing is continueduntil a maximum of D-pantothenic acid has formed. This target is usuallyreached within 10 hours to 160 hours.

[0105] The D-pantothenic acid or the corresponding salts ofD-pantothenic acid contained in the fermentation broth can be isolatedand purified in accordance with known methods.

[0106] It is also possible for the fermentation broths comprisingD-pantothenic acid and/or salts thereof preferably first to be freedfrom all or some of the biomass by known separation methods, such as,for example, centrifugation, filtration, decanting or a combinationthereof. However, it is also possible to leave the biomass in itsentirety in the fermentation broth. In general, the suspension orsolution is preferably concentrated and worked up to a powder, forexample with the aid of a spray dryer or a freeze-drying unit. Thispowder is then in general converted by suitable compacting orgranulating processes, e.g. also build-up granulation, into acoarser-grained, free-flowing, storable and largely dust-free productwith the desired particle size distribution of optionally 20 to 2000 μm.In the granulation or compacting it is advantageous to employconventional organic or inorganic auxiliary substances or carriers, suchas starch, gelatin, cellulose derivatives or similar substances, such asare conventionally used as binders, gelling agents or thickeners infoodstuffs or feedstuffs processing, or further substances, such as, forexample, silicas, silicates or stearates.

[0107] Alternatively, the fermentation product, with or without furtherconventional fermentation constituents, can be absorbed on to an organicor inorganic carrier substance which is known and conventional infeedstuffs processing, such as, for example, silicas, silicates, grits,brans, meals, starches, sugars or others, and/or stabilized withconventional thickeners or binders. Use examples and processes in thiscontext are described in the literature (Die Mühle+Mischfuttertechnik132 (1995) 49, page 817). D-Pantothenic acid, or the desired salt ofD-pantothenic acid or a formulation comprising these compounds, isoptionally added at a suitable process stage in order to achieve orestablish the desired content of pantothenic acid, pantothenic acidderivative, or of the desired salt in the end product.

[0108] The desired content is in general in the range from 20 to 80 wt.% (dry mass).

[0109] The concentration of pantothenic acid can be determined withknown chemical (Velisek; Chromatographic Science 60, 515-560 (1992)) ormicrobiological methods, such as e.g. the Lactobacillus plantarum test(DIFCO MANUAL, 10^(th) Edition, p. 1100-1102; Michigan, USA).

[0110] Products, such as feedstuffs or feedstuff additives, obtained bythe concentration of the culture medium, fermentation medium, orfermentive microorganisms may be further supplemented with othernutritional products, such as minerals or vitamins. For instance,pantothenic acid synergists such as vitamin B12 may be added. Othersubstances that facilitate the uptake or utilization of pantothenicacid, such as beta-carotene, vitamin A, vitamin C, vitamin E, vitaminB6, folic acid or biotin may also be added. Moreover, the pH of suchproducts may be adjusted using convention acids, bases or buffers toenhance the stability of the pantothenic acid, its salt or derivative.

[0111] Such feedstuffs or feedstuff additives may be administered tolivestock animals, such as bovines, pigs, goats, sheep, horses, camels,and llamas; avians, such as chickens, geese or ducks; pets or domesticanimals, such as cat and dogs, birds or tropical fish; laboratoryanimals, such as mice, rats and hamsters; wild herbiferous, omnivorousor carnivorous animals such reptiles, zebras, elephants, bears, lionsand tigers; or may be added to aquaculture fees, such as those used forfish or crustaceans; or to products used to feed insects, such as bees.

[0112] A pure culture of the Escherichia coli K-12 strain DH5α/pMAK705was deposited as DSM 13720 on 8^(th) September 2000 at the DeutscheSammlung fur Mikroorganismen und Zellkulturen (DSMZ=German Collection ofMicroorganisms and Cell Cultures, Braunschweig, Germany) in accordancewith the Budapest Treaty.

[0113] A pure culture of the Escherichia coli K-12 strain MG442ΔpckA wasdeposited as DSM 13761 on Oct. 2, 2000 at the Deutsche Sammlung fürMikroorganismen und Zellkulturen (DSMZ=German Collection ofMicroorganisms and Cell Cultures, Braunschweig, Germany) in accordancewith the Budapest Treaty.

[0114] The present invention is explained in more detail in thefollowing with the aid of embodiment examples.

[0115] The isolation of plasmid DNA from Escherichia coli and alltechniques of restriction, Klenow and alkaline phosphatase treatment arecarried out by the method of Sambrook et al. (Molecular cloning—ALaboratory Manual, Cold Spring Harbor Laboratory Press (1989). Unlessdescribed otherwise, the transformation of Escherichia coli is carriedout by the method of Chung et al., Proceedings of the National Academyof Sciences of the United States of America USA 86: 2172-2175 (1989).

[0116] The incubation temperature for the preparation of strains andtransformants is 37° C. Temperatures of 30° C. and 44° C. are used inthe gene replacement method of Hamilton et.al. ibid. (1989).

EXAMPLE 1

[0117] Construction of the deletion mutation of the pckA gene

[0118] Parts of the 5′ and 3′ region of the pckA gene are amplified fromEscherichia coli K12 using the polymerase chain reaction (PCR) andsynthetic oligonucleotides. Starting from the nucleotide sequence of thepckA gene in

[0119]E. coli K12 MG1655 (SEQ ID No. 1), the following PCR primers aresynthesized (MWG Biotech, Ebersberg, Germany):

[0120] pckA′5′-1: 5′- GATCCGAGCCTGACAGGTTA -3′ (SEQ ID No. 3)

[0121] pckA′5′-2: 5′- GCATGCGCTCGGTCAGGTTA -3′ (SEQ ID No. 4)

[0122] pckA′3′-1: 5′- AGGCCTGAAGATGGCACTATCG -3′ (SEQ ID No. 5)

[0123] pckA′3′-2: 5′- CCGGAGAAGCGTAGGTGTTA -3′ (SEQ ID No. 6)

[0124] The chromosomal E. coli K12 MG1655 DNA employed for the PCR isisolated according to the manufacturers instructions with “QiagenGenomic-tips 100/G” (QIAGEN, Hilden, Germany). A DNA fragment approx.500 bp in size from the 5′ region of the pckA gene (called pck1) and aDNA fragment approx. 600 bp in size from the 3′ region of the pckA gene(called pck2) can be amplified with the specific primers under standardPCR conditions (Innis et al. (1990) PCR Protocols. A Guide to Methodsand Applications, Academic Press) with Taq-DNA polymerase (Gibco-BRL,Eggenstein, Germany). The PCR products are each ligated with the vectorpCR2.1TOPO (TOPO TA Cloning Kit, Invitrogen, Groningen, The Netherlands)in accordance with the manufacturers instructions and transformed intothe E. coli strain TOP10F′.

[0125] Selection of plasmid-carrying cells takes place on LB agar, towhich 50 μg/ml ampicillin are added. After isolation of the plasmid DNA,the vector pCR2.1TOPOpck2 is cleaved with the restriction enzymes StuIand XbaI and, after separation in 0.8% agarose gel, the pck2 fragment isisolated with the aid of the QIAquick Gel Extraction Kit (QIAGEN,Hilden, Germany). After isolation of the plasmid DNA the vectorpCR2.1TOPOpck1 is cleaved with the enzymes EcoRV and XbaI and ligatedwith the pck2 fragment isolated. The E. coli strain DH5α is transformedwith the ligation batch and plasmid-carrying cells are selected on LBagar, to which 50 μg/ml ampicillin is added. After isolation of theplasmid DNA those plasmids in which the mutagenic DNA sequence shown inSEQ ID No. 7 is cloned are detected by control cleavage with the enzymesSpeI and XbaI. One of the plasmids is called pCR2.1TOPOΔpckA.

EXAMPLE 2

[0126] Construction of the replacement vector pMAK705ΔpckA

[0127] The pckA allele described in example 1 is isolated from thevector pCR2.1TOPOΔpckA after restriction with the enzymes SpeI and XbaIand separation in 0.8% agarose gel, and ligated with the plasmid pMAK705(Hamilton et al. (1989) Journal of Bacteriology 174, 4617-4622), whichhas been digested with the enzyme XbaI. The ligation batch istransformed in DH5α and plasmid-carrying cells are selected on LB agar,to which 20 μg/ml chloramphenicol are added. Successful cloning isdemonstrated after isolation of the plasmid DNA and cleavage with theenzymes HpaI, KpnI, HindIII, SalI and PstI. The replacement vectorformed, pMAK705ΔpckA (=pMAK705deltapckA), is shown in FIG. 1.

EXAMPLE 3

[0128] Position-specific mutagenesis of the pckA gene in the E. colistrain MG442

[0129] The L-threonine-producing E. coli strain MG442 is described inthe patent specification U.S. Pat. No. 4,278,765 and deposited as CMIMB-1628 at the Russian National Collection for Industrial Microorganisms(VKPM, Moscow, Russia).

[0130] For replacement of the chromosomal pckA gene with theplasmid-coded deletion construct, MG442 is transformed with the plasmidpMAK705ΔpckA, The gene replacement is carried out by the selectionmethod described by Hamilton et al. (1989) Journal of Bacteriology 174,4617-4622) and is verified by standard PCR methods (Innis et al. (1990)PCR Protocols. A Guide to Methods and Applications, Academic Press) withthe following oligonucleotide primers:

[0131] pckA′5′-1: 5′- GATCCGAGCCTGACAGGTTA -3′ (SEQ ID No. 3)

[0132] pckA′3′-2: 5′- CCGGAGAAGCGTAGGTGTTA -3′ (SEQ ID No. 6)

[0133] After replacement has taken place, MG442 contains the form of theΔpckA allele shown in SEQ ID No. 8. The strain obtained is calledMG442ΔpckA.

EXAMPLE 4

[0134] Preparation of D-pantothenic acid with the strainMG442ΔpckA/pFV3lilvGM

[0135] 4.1 Amplification and Cloning of the ilvGM Gene

[0136] The ilvGM operon from Escherichia coli IF03547 which codes foracetohydroxy acid synthase II (Institut für Fermentation [Institute ofFermentation], Osaka, Japan) is amplified using the polymerase chainreaction (PCR) and synthetic oligonucleotides. Starting from thenucleotide sequence of the ilvGM operon in E. coli K12 MG1655 (GenBank:Accession No. M87049), PCR primers are synthesized, (MWG Biotech,Ebersberg, Germany). The sequence of the primer ilvGM1 is chosen suchthat it contains an adenine at position 8. As a result, a modifiedribosome binding site is generated 7 nucleotides upstream of the startcodon of the ilvG protein.

[0137] IlvGM1: 5′-CAGGACGAGGAACTAACTATG -3′ (SEQ ID No. 9)

[0138] IlvGM2: 5′-TCACGATGGCGGAATACAAC -3′ (SEQ ID No. 10)

[0139] The chromosomal E. coli IF03547 DNA employed for the PCR isisolated according to the manufacturers instructions with “QIAGENGenomic-tips 100/G” (QIAGEN, Hilden, Germany). A DNA fragment approx.2100 bp in size, which comprises the modified ribosome binding site, theilvGM coding regions and approx. 180 bp 3′-flanking sequences, can beamplified with the specific primers under standard PCR conditions (Inniset al.: PCR Protocols. A Guide to Methods and Applications, 1990,Academic Press) with Pfu-DNA polymerase (Promega Corporation, Madison,USA). The PCR product is cloned in the plasmid pCR-BluntII-TOPO andtransformed in the E. coli strain TOP10 (Invitrogen, Groningen, TheNetherlands, Product Description Zero Blunt TOPO PCR Cloning Kit, Cat.No. K2800-20). Successful cloning is demonstrated by cleavage of theplasmid pCR-BluntIFO3547ilvGM with the restriction enzymes EcoRI andSphI. For this, the plasmid DNA is isolated by means of the “QIAprepSpin Plasmid Kit” (QIAGEN, Hilden, Germany) and, after cleavage,separated in a 0.8% agarose gel. The DNA sequence of the amplifiedfragment is determined using the reverse and universal sequencing primer(QIAGEN, Hilden, Germany). The sequence of the PCR product is shown inSEQ ID No. 11 and 13. The ilvG gene or allele is identified in SEQ IDNo. 11. The ilvM gene or allele is identified in SEQ ID No. 13. Theassociated gene products or proteins are shown in SEQ ID No. 12 and 14.

[0140] 4.2 Cloning of the ilvGM Gene in the Expression Vector pTrc99A

[0141] The ilvGM genes described in example 4.1 are cloned in the vectorpTrc99A (Amersham Pharmacia Biotech Inc, Uppsala, Sweden) for expressionin Escherichia coli K12. For this, the plasmid pCR-BluntIFO3547ilvGM iscleaved with the enzyme EcoRI, the cleavage batch is separated in 0.8%agarose gel and the ilvGM fragment 2.1 kbp in size is isolated with theaid of the QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany). Thevector pTrc99A is cleaved with the enzyme EcoRI, an alkaline phosphatasetreatment is carried out, and ligation is carried out with the ilvGMfragment isolated. The ligation batch is transformed in the E. colistrain DH5A. Selection of pTrc99A-carrying cells is carried out on LBagar (Lennox, Virology 1:190 (1955)), to which 50 μg/ml ampicillin isadded. Successful cloning of the ilvGM operon can be demonstrated afterplasmid DNA isolation and control cleavage with SalI and SphI. In thevector, which is called pTrc99AilvGM (FIG. 2), expression of the ilvGMoperon is regulated by the Ptrc promoter lying upstream of the modifiedribosome binding site and by the rRNA terminator region lying downstreamof the ilvGM coding region.

[0142] 4.3 Construction of the Vector pFV31ilvGM

[0143] The E. coli strain FV5069/pFV31 which produces D-pantothenic acidis described in EP-A-0590857 and deposited as FERM BP 4395 in accordancewith the Budapest Treaty. The plasmid pFV31 is isolated fromFV5069/pFV31, cleaved with the enzyme BamHI, and the projecting 3′ endsare treated with Klenow enzyme. An alkaline phosphatase treatment isthen carried out. From the vector pTrc99AilvGM described in example 4.2,after restriction with the enzyme SspI and separation of the cleavagebatch in 0.8% agarose gel, the ilvGM expression cassette 2.8 kbp in sizeis isolated and ligated with the linearized and dephosphorylated vectorpFV31. The ligation batch is transformed in the E. coli strain DH5α andplasmid-carrying cells are selected on LB agar, to which 50 μg/mlampicillin are added. Successful cloning of the ilvGM expressioncassette can be demonstrated after plasmid DNA isolation and controlcleavage with HindIII, SalI, SmaI, SphI and XbaI. The plasmid is calledpFV31ilvGM (FIG. 3).

[0144] 4.4 Preparation of the Strain MG442ΔpckA/pFV3lilvGM

[0145] The strain MG442ΔpckA obtained in example 3 and the strain MG442are transformed with the plasmid pFV31ilvGM and transformants areselected on LB agar, which is supplemented with 50 μg/ml ampicillin. Thestrains MG442ΔpckA/pFV31ilvGM and MG442/pFV31ilvGM are formed in thismanner.

[0146] 4.5 Preparation of D-pantothenic Acid with the StrainMG442ΔpckA/pFV31ilvGM

[0147] The pantothenate production of the E. coli strainsMG442/pFV31ilvGM and MG442ΔpckA/pFV31ilvGM is checked in batch culturesof 10 ml contained in 100 ml conical flasks. For this, 10 ml ofpreculture medium of the following composition: 2 g/l yeast extract, 10g/l (NH₄)₂SO₄, 1 g/l KH₂PO₄, 0.5 g/l MgSO₄*7H₂O, 15 g/l CaCO₃, 20/1glucose, 50 μg/ml ampicillin, are inoculated with an individual colonyand incubated for 20 hours at 33° C. and 200 rpm on an ESR incubatorfrom Kühner AG (Birsfelden, Switzerland). In each case 200 μl of thispreculture are transinoculated into 10 ml of production medium (25 g/l(NH₄)₂SO₄, 2 g/l KH₂PO₄, 1 g/l MgSO₄*7H₂O, 0.03 g/l FeSO₄*7H₂O, 0.018g/l MnSO₄*1H₂O, 30 g/l CaCO₃, 20 g/l glucose, 20 g/l β-alanine, 250 mg/lthiamine) and the batch is incubated for 48 hours at 37° C. and 200 rpm.After the incubation the optical density (OD) of the culture suspensionis determined with an LP2W photometer from Dr. Lange (Düsseldorf,Germany) at a measurement wavelength of 660 nm.

[0148] The concentration of D-pantothenate formed in thesterile-filtered culture supernatant is then determined by means of theLactobacillus plantarum ATCC8014 pantothenate assay in accordance withthe instructions of DIFCO, DIFCO MANUAL, 10^(th) Edition, p. 1100-1102;Michigan, USA. D(+)-Pantothenic acid calcium salt hydrate (cataloguenumber 25,972-1, Sigma-Aldrich, Deisenhofen, Germany) is used for thecalibration.

[0149] The result of the experiment is shown in table 1. TABLE 1 ODPantothenate Strain (660 nm) g/l MG442/pFV31ilvGM 2.7 1.35 MG442ΔpckA/3.5 1.85 pFV31ilvGM

[0150] Modifications and Other Embodiments

[0151] Various modifications and variations of the described nucleicacids, plasmids, and cells as well as compositions and methods of usingsuch products and the concept of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed is not intended to be limited to such specificembodiments. Various modifications of the described modes for carryingout the invention which are obvious to those skilled in the molecularbiological, biochemical, chemical, chemical engineering, medical, orpharmacological arts or related fields are intended to be within thescope of the following claims.

[0152] Incorporation by Reference

[0153] Each document, patent application or patent publication cited byor referred to in this disclosure is incorporated by reference in itsentirety. Any patent document to which this application claims priorityis also incorporated by reference in its entirety. Specifically,priority documents DE 101 12 100.8, filed Mar. 14, 2001 and U.S.Provisional Application 60/304,774, filed Jul. 13, 2001 are herebyincorporated by reference.

1 14 1 1623 DNA Escherichia coli CDS (1)..(1620) pckA 1 atg cgc gtt aacaat ggt ttg acc ccg caa gaa ctc gag gct tat ggt 48 Met Arg Val Asn AsnGly Leu Thr Pro Gln Glu Leu Glu Ala Tyr Gly 1 5 10 15 atc agt gac gtacat gat atc gtt tac aac cca agc tac gac ctg ctg 96 Ile Ser Asp Val HisAsp Ile Val Tyr Asn Pro Ser Tyr Asp Leu Leu 20 25 30 tat cag gaa gag ctcgat ccg agc ctg aca ggt tat gag cgc ggg gtg 144 Tyr Gln Glu Glu Leu AspPro Ser Leu Thr Gly Tyr Glu Arg Gly Val 35 40 45 tta act aat ctg ggt gccgtt gcc gtc gat acc ggg atc ttc acc ggt 192 Leu Thr Asn Leu Gly Ala ValAla Val Asp Thr Gly Ile Phe Thr Gly 50 55 60 cgt tca cca aaa gat aag tatatc gtc cgt gac gat acc act cgc gat 240 Arg Ser Pro Lys Asp Lys Tyr IleVal Arg Asp Asp Thr Thr Arg Asp 65 70 75 80 act ttc tgg tgg gca gac aaaggc aaa ggt aag aac gac aac aaa cct 288 Thr Phe Trp Trp Ala Asp Lys GlyLys Gly Lys Asn Asp Asn Lys Pro 85 90 95 ctc tct ccg gaa acc tgg cag catctg aaa ggc ctg gtg acc agg cag 336 Leu Ser Pro Glu Thr Trp Gln His LeuLys Gly Leu Val Thr Arg Gln 100 105 110 ctt tcc ggc aaa cgt ctg ttc gttgtc gac gct ttc tgt ggt gcg aac 384 Leu Ser Gly Lys Arg Leu Phe Val ValAsp Ala Phe Cys Gly Ala Asn 115 120 125 ccg gat act cgt ctt tcc gtc cgtttc atc acc gaa gtg gcc tgg cag 432 Pro Asp Thr Arg Leu Ser Val Arg PheIle Thr Glu Val Ala Trp Gln 130 135 140 gcg cat ttt gtc aaa aac atg tttatt cgc ccg agc gat gaa gaa ctg 480 Ala His Phe Val Lys Asn Met Phe IleArg Pro Ser Asp Glu Glu Leu 145 150 155 160 gca ggt ttc aaa cca gac tttatc gtt atg aac ggc gcg aag tgc act 528 Ala Gly Phe Lys Pro Asp Phe IleVal Met Asn Gly Ala Lys Cys Thr 165 170 175 aac ccg cag tgg aaa gaa cagggt ctc aac tcc gaa aac ttc gtg gcg 576 Asn Pro Gln Trp Lys Glu Gln GlyLeu Asn Ser Glu Asn Phe Val Ala 180 185 190 ttt aac ctg acc gag cgc atgcag ctg att ggc ggc acc tgg tac ggc 624 Phe Asn Leu Thr Glu Arg Met GlnLeu Ile Gly Gly Thr Trp Tyr Gly 195 200 205 ggc gaa atg aag aaa ggg atgttc tcg atg atg aac tac ctg ctg ccg 672 Gly Glu Met Lys Lys Gly Met PheSer Met Met Asn Tyr Leu Leu Pro 210 215 220 ctg aaa ggt atc gct tct atgcac tgc tcc gcc aac gtt ggt gag aaa 720 Leu Lys Gly Ile Ala Ser Met HisCys Ser Ala Asn Val Gly Glu Lys 225 230 235 240 ggc gat gtt gcg gtg ttcttc ggc ctt tcc ggc acc ggt aaa acc acc 768 Gly Asp Val Ala Val Phe PheGly Leu Ser Gly Thr Gly Lys Thr Thr 245 250 255 ctt tcc acc gac ccg aaacgt cgc ctg att ggc gat gac gaa cac ggc 816 Leu Ser Thr Asp Pro Lys ArgArg Leu Ile Gly Asp Asp Glu His Gly 260 265 270 tgg gac gat gac ggc gtgttt aac ttc gaa ggc ggc tgc tac gca aaa 864 Trp Asp Asp Asp Gly Val PheAsn Phe Glu Gly Gly Cys Tyr Ala Lys 275 280 285 act atc aag ctg tcg aaagaa gcg gaa cct gaa atc tac aac gct atc 912 Thr Ile Lys Leu Ser Lys GluAla Glu Pro Glu Ile Tyr Asn Ala Ile 290 295 300 cgt cgt gat gcg ttg ctggaa aac gtc acc gtg cgt gaa gat ggc act 960 Arg Arg Asp Ala Leu Leu GluAsn Val Thr Val Arg Glu Asp Gly Thr 305 310 315 320 atc gac ttt gat gatggt tca aaa acc gag aac acc cgc gtt tct tat 1008 Ile Asp Phe Asp Asp GlySer Lys Thr Glu Asn Thr Arg Val Ser Tyr 325 330 335 ccg atc tat cac atcgat aac att gtt aag ccg gtt tcc aaa gcg ggc 1056 Pro Ile Tyr His Ile AspAsn Ile Val Lys Pro Val Ser Lys Ala Gly 340 345 350 cac gcg act aag gttatc ttc ctg act gct gat gct ttc ggc gtg ttg 1104 His Ala Thr Lys Val IlePhe Leu Thr Ala Asp Ala Phe Gly Val Leu 355 360 365 ccg ccg gtt tct cgcctg act gcc gat caa acc cag tat cac ttc ctc 1152 Pro Pro Val Ser Arg LeuThr Ala Asp Gln Thr Gln Tyr His Phe Leu 370 375 380 tct ggc ttc acc gccaaa ctg gcc ggt act gag cgt ggc atc acc gaa 1200 Ser Gly Phe Thr Ala LysLeu Ala Gly Thr Glu Arg Gly Ile Thr Glu 385 390 395 400 ccg acg cca accttc tcc gct tgc ttc ggc gcg gca ttc ctg tcg ctg 1248 Pro Thr Pro Thr PheSer Ala Cys Phe Gly Ala Ala Phe Leu Ser Leu 405 410 415 cac ccg act cagtac gca gaa gtg ctg gtg aaa cgt atg cag gcg gcg 1296 His Pro Thr Gln TyrAla Glu Val Leu Val Lys Arg Met Gln Ala Ala 420 425 430 ggc gcg cag gcttat ctg gtt aac act ggc tgg aac ggc act ggc aaa 1344 Gly Ala Gln Ala TyrLeu Val Asn Thr Gly Trp Asn Gly Thr Gly Lys 435 440 445 cgt atc tcg attaaa gat acc cgc gcc att atc gac gcc atc ctc aac 1392 Arg Ile Ser Ile LysAsp Thr Arg Ala Ile Ile Asp Ala Ile Leu Asn 450 455 460 ggt tcg ctg gataat gca gaa acc ttc act ctg ccg atg ttt aac ctg 1440 Gly Ser Leu Asp AsnAla Glu Thr Phe Thr Leu Pro Met Phe Asn Leu 465 470 475 480 gcg atc ccaacc gaa ctg ccg ggc gta gac acg aag att ctc gat ccg 1488 Ala Ile Pro ThrGlu Leu Pro Gly Val Asp Thr Lys Ile Leu Asp Pro 485 490 495 cgt aac acctac gct tct ccg gaa cag tgg cag gaa aaa gcc gaa acc 1536 Arg Asn Thr TyrAla Ser Pro Glu Gln Trp Gln Glu Lys Ala Glu Thr 500 505 510 ctg gcg aaactg ttt atc gac aac ttc gat aaa tac acc gac acc cct 1584 Leu Ala Lys LeuPhe Ile Asp Asn Phe Asp Lys Tyr Thr Asp Thr Pro 515 520 525 gcg ggt gccgcg ctg gta gcg gct ggt ccg aaa ctg taa 1623 Ala Gly Ala Ala Leu Val AlaAla Gly Pro Lys Leu 530 535 540 2 540 PRT Escherichia coli 2 Met Arg ValAsn Asn Gly Leu Thr Pro Gln Glu Leu Glu Ala Tyr Gly 1 5 10 15 Ile SerAsp Val His Asp Ile Val Tyr Asn Pro Ser Tyr Asp Leu Leu 20 25 30 Tyr GlnGlu Glu Leu Asp Pro Ser Leu Thr Gly Tyr Glu Arg Gly Val 35 40 45 Leu ThrAsn Leu Gly Ala Val Ala Val Asp Thr Gly Ile Phe Thr Gly 50 55 60 Arg SerPro Lys Asp Lys Tyr Ile Val Arg Asp Asp Thr Thr Arg Asp 65 70 75 80 ThrPhe Trp Trp Ala Asp Lys Gly Lys Gly Lys Asn Asp Asn Lys Pro 85 90 95 LeuSer Pro Glu Thr Trp Gln His Leu Lys Gly Leu Val Thr Arg Gln 100 105 110Leu Ser Gly Lys Arg Leu Phe Val Val Asp Ala Phe Cys Gly Ala Asn 115 120125 Pro Asp Thr Arg Leu Ser Val Arg Phe Ile Thr Glu Val Ala Trp Gln 130135 140 Ala His Phe Val Lys Asn Met Phe Ile Arg Pro Ser Asp Glu Glu Leu145 150 155 160 Ala Gly Phe Lys Pro Asp Phe Ile Val Met Asn Gly Ala LysCys Thr 165 170 175 Asn Pro Gln Trp Lys Glu Gln Gly Leu Asn Ser Glu AsnPhe Val Ala 180 185 190 Phe Asn Leu Thr Glu Arg Met Gln Leu Ile Gly GlyThr Trp Tyr Gly 195 200 205 Gly Glu Met Lys Lys Gly Met Phe Ser Met MetAsn Tyr Leu Leu Pro 210 215 220 Leu Lys Gly Ile Ala Ser Met His Cys SerAla Asn Val Gly Glu Lys 225 230 235 240 Gly Asp Val Ala Val Phe Phe GlyLeu Ser Gly Thr Gly Lys Thr Thr 245 250 255 Leu Ser Thr Asp Pro Lys ArgArg Leu Ile Gly Asp Asp Glu His Gly 260 265 270 Trp Asp Asp Asp Gly ValPhe Asn Phe Glu Gly Gly Cys Tyr Ala Lys 275 280 285 Thr Ile Lys Leu SerLys Glu Ala Glu Pro Glu Ile Tyr Asn Ala Ile 290 295 300 Arg Arg Asp AlaLeu Leu Glu Asn Val Thr Val Arg Glu Asp Gly Thr 305 310 315 320 Ile AspPhe Asp Asp Gly Ser Lys Thr Glu Asn Thr Arg Val Ser Tyr 325 330 335 ProIle Tyr His Ile Asp Asn Ile Val Lys Pro Val Ser Lys Ala Gly 340 345 350His Ala Thr Lys Val Ile Phe Leu Thr Ala Asp Ala Phe Gly Val Leu 355 360365 Pro Pro Val Ser Arg Leu Thr Ala Asp Gln Thr Gln Tyr His Phe Leu 370375 380 Ser Gly Phe Thr Ala Lys Leu Ala Gly Thr Glu Arg Gly Ile Thr Glu385 390 395 400 Pro Thr Pro Thr Phe Ser Ala Cys Phe Gly Ala Ala Phe LeuSer Leu 405 410 415 His Pro Thr Gln Tyr Ala Glu Val Leu Val Lys Arg MetGln Ala Ala 420 425 430 Gly Ala Gln Ala Tyr Leu Val Asn Thr Gly Trp AsnGly Thr Gly Lys 435 440 445 Arg Ile Ser Ile Lys Asp Thr Arg Ala Ile IleAsp Ala Ile Leu Asn 450 455 460 Gly Ser Leu Asp Asn Ala Glu Thr Phe ThrLeu Pro Met Phe Asn Leu 465 470 475 480 Ala Ile Pro Thr Glu Leu Pro GlyVal Asp Thr Lys Ile Leu Asp Pro 485 490 495 Arg Asn Thr Tyr Ala Ser ProGlu Gln Trp Gln Glu Lys Ala Glu Thr 500 505 510 Leu Ala Lys Leu Phe IleAsp Asn Phe Asp Lys Tyr Thr Asp Thr Pro 515 520 525 Ala Gly Ala Ala LeuVal Ala Ala Gly Pro Lys Leu 530 535 540 3 20 DNA artificial sequencesynthetic DNA 3 gatccgagcc tgacaggtta 20 4 20 DNA artificial sequencesynthetic DNA 4 gcatgcgctc ggtcaggtta 20 5 22 DNA artificial sequencesynthetic DNA 5 aggcctgaag atggcactat cg 22 6 20 DNA artificial sequencesynthetic DNA 6 ccggagaagc gtaggtgtta 20 7 1156 DNA Escherichia colimisc (1)..(1156) Mutagene DNA 7 ctagtaacgg ccgccagtgt gctggaattcggcttgatcc gagcctgaca ggttatgagc 60 gcggggtgtt aactaatctg ggtgccgttgccgtcgatac cgggatcttc accggtcgtt 120 caccaaaaga taagtatatc gtccgtgacgataccactcg cgatactttc tggtgggcag 180 acaaaggcaa aggtaagaac gacaacaaacctctctctcc ggaaacctgg cagcatctga 240 aaggcctggt gaccaggcag ctttccggcaaacgtctgtt cgttgtcgac gctttctgtg 300 gtgcgaaccc ggatactcgt ctttccgtccgtttcatcac cgaagtggcc tggcaggcgc 360 attttgtcaa aaacatgttt attcgcccgagcgatgaaga actggcaggt ttcaaaccag 420 actttatcgt tatgaacggc gcgaagtgcactaacccgca gtggaaagaa cagggtctca 480 actccgaaaa cttcgtggcg tttaacctgaccgagcgcat gcaagccgaa ttctgcagat 540 cctgaagatg gcactatcga ctttgatgatggttcaaaaa ccgagaacac ccgcgtttct 600 tatccgatct atcacatcga taacattgttaagccggttt ccaaagcggg ccacgcgact 660 aaggttatct tcctgactgc tgatgctttcggcgtgttgc cgccggtttc tcgcctgact 720 gccgatcaaa cccagtatca cttcctctctggcttcaccg ccaaactggc cggtactgag 780 cgtggcatca ccgaaccgac gccaaccttctccgcttgct tcggcgcggc attcctgtcg 840 ctgcacccga ctcagtacgc agaagtgctggtgaaacgta tgcaggcggc gggcgcgcag 900 gcttatctgg ttaacactgg ctggaacggcactggcaaac gtatctcgat taaagatacc 960 cgcgccatta tcgacgccat cctcaacggttcgctggata atgcagaaac cttcactctg 1020 ccgatgttta acctggcgat cccaaccgaactgccgggcg tagacacgaa gattctcgat 1080 ccgcgtaaca cctacgcttc tccggaagccgaattctgca gatatccatc acactggcgg 1140 ccgctcgagc atgcat 1156 8 1294 DNAEscherichia coli misc (1)..(3) Start codon of delta pckA allele 8atgcgcgtta acaatggttt gaccccgcaa gaactcgagg cttatggtat cagtgacgta 60catgatatcg tttacaaccc aagctacgac ctgctgtatc aggaagagct cgatccgagc 120ctgacaggtt atgagcgcgg ggtgttaact aatctgggtg ccgttgccgt cgataccggg 180atcttcaccg gtcgttcacc aaaagataag tatatcgtcc gtgacgatac cactcgcgat 240actttctggt gggcagacaa aggcaaaggt aagaacgaca acaaacctct ctctccggaa 300acctggcagc atctgaaagg cctggtgacc aggcagcttt ccggcaaacg tctgttcgtt 360gtcgacgctt tctgtggtgc gaacccggat actcgtcttt ccgtccgttt catcaccgaa 420gtggcctggc aggcgcattt tgtcaaaaac atgtttattc gcccgagcga tgaagaactg 480gcaggtttca aaccagactt tatcgttatg aacggcgcga agtgcactaa cccgcagtgg 540aaagaacagg gtctcaactc cgaaaacttc gtggcgttta acctgaccga gcgcatgcaa 600gccgaattct gcagatcctg aagatggcac tatcgacttt gatgatggtt caaaaaccga 660gaacacccgc gtttcttatc cgatctatca catcgataac attgttaagc cggtttccaa 720agcgggccac gcgactaagg ttatcttcct gactgctgat gctttcggcg tgttgccgcc 780ggtttctcgc ctgactgccg atcaaaccca gtatcacttc ctctctggct tcaccgccaa 840actggccggt actgagcgtg gcatcaccga accgacgcca accttctccg cttgcttcgg 900cgcggcattc ctgtcgctgc acccgactca gtacgcagaa gtgctggtga aacgtatgca 960ggcggcgggc gcgcaggctt atctggttaa cactggctgg aacggcactg gcaaacgtat 1020ctcgattaaa gatacccgcg ccattatcga cgccatcctc aacggttcgc tggataatgc 1080agaaaccttc actctgccga tgtttaacct ggcgatccca accgaactgc cgggcgtaga 1140cacgaagatt ctcgatccgc gtaacaccta cgcttctccg gaacagtggc aggaaaaagc 1200cgaaaccctg gcgaaactgt ttatcgacaa cttcgataaa tacaccgaca cccctgcggg 1260tgccgcgctg gtagcggctg gtccgaaact gtaa 1294 9 21 DNA artificial sequencesynthetic DNA 9 caggacgagg aactaactat g 21 10 20 DNA artificial sequencesynthetic DNA 10 tcacgatggc ggaatacaac 20 11 2111 DNA Escherichia coliRBS (8)..(12) 11 caggacgagg aactaact atg aat ggc gca cag tgg gtg gta catgcg ttg 51 Met Asn Gly Ala Gln Trp Val Val His Ala Leu 1 5 10 cgg gcacag ggt gtg aac acc gtt ttc ggt tat ccg ggt ggc gca att 99 Arg Ala GlnGly Val Asn Thr Val Phe Gly Tyr Pro Gly Gly Ala Ile 15 20 25 atg ccg gtttac gat gca ttg tat gac ggc ggc gtg gag cac ttg ctg 147 Met Pro Val TyrAsp Ala Leu Tyr Asp Gly Gly Val Glu His Leu Leu 30 35 40 tgc cga cat gagcag ggt gcg gca atg gcg gct atc ggt tat gcc cgt 195 Cys Arg His Glu GlnGly Ala Ala Met Ala Ala Ile Gly Tyr Ala Arg 45 50 55 gct acc ggc aaa actggc gta tgt atc gcc acg tct ggt ccg ggc gca 243 Ala Thr Gly Lys Thr GlyVal Cys Ile Ala Thr Ser Gly Pro Gly Ala 60 65 70 75 acc aac ctg ata accggg ctt gcg gac gca ctg tta gat tct atc cct 291 Thr Asn Leu Ile Thr GlyLeu Ala Asp Ala Leu Leu Asp Ser Ile Pro 80 85 90 gtt gtt gcc atc acc ggtcaa gtg tcc gca ccg ttt atc ggc acg gac 339 Val Val Ala Ile Thr Gly GlnVal Ser Ala Pro Phe Ile Gly Thr Asp 95 100 105 gca ttt cag gaa gtg gatgtc ctg gga ttg tcg tta gcc tgt acc aag 387 Ala Phe Gln Glu Val Asp ValLeu Gly Leu Ser Leu Ala Cys Thr Lys 110 115 120 cac agc ttt ctg gtg cagtcg ctg gaa gag ttg ccg cgc att atg gct 435 His Ser Phe Leu Val Gln SerLeu Glu Glu Leu Pro Arg Ile Met Ala 125 130 135 gaa gca ttc gac gtt gccagc tca ggt cgt cct ggt ccg gtt ctg gtc 483 Glu Ala Phe Asp Val Ala SerSer Gly Arg Pro Gly Pro Val Leu Val 140 145 150 155 gat atc cca aaa gatatc cag cta gcc agc ggt gac ctg gaa ccg tgg 531 Asp Ile Pro Lys Asp IleGln Leu Ala Ser Gly Asp Leu Glu Pro Trp 160 165 170 ttc acc acc gtt gaaaac gaa gtg act ttc cca cat gcc gaa gtt gag 579 Phe Thr Thr Val Glu AsnGlu Val Thr Phe Pro His Ala Glu Val Glu 175 180 185 caa gcg cgc cag atgctg gca aaa gcg caa aaa ccg atg ctg tac gtt 627 Gln Ala Arg Gln Met LeuAla Lys Ala Gln Lys Pro Met Leu Tyr Val 190 195 200 ggt ggt ggc gtg ggtatg gcg cag gca gtt cct gct tta cga gaa ttt 675 Gly Gly Gly Val Gly MetAla Gln Ala Val Pro Ala Leu Arg Glu Phe 205 210 215 ctc gct acc aca aaaatg cct gcc acc tgc acg ctg aaa ggg ctg ggc 723 Leu Ala Thr Thr Lys MetPro Ala Thr Cys Thr Leu Lys Gly Leu Gly 220 225 230 235 gca gtt gaa gcagat tat ccg tac tat ctg ggc atg ctg gga atg cat 771 Ala Val Glu Ala AspTyr Pro Tyr Tyr Leu Gly Met Leu Gly Met His 240 245 250 ggc acc aaa gcggcg aac ttc gcg gtg cag gag tgc gac ttg ctg atc 819 Gly Thr Lys Ala AlaAsn Phe Ala Val Gln Glu Cys Asp Leu Leu Ile 255 260 265 gcc gtg ggt gcacgt ttt gat gac cgg gtg acc ggc aaa ctg aac acc 867 Ala Val Gly Ala ArgPhe Asp Asp Arg Val Thr Gly Lys Leu Asn Thr 270 275 280 ttc gca cca cacgcc agt gtt atc cat atg gat atc gac ccg gca gaa 915 Phe Ala Pro His AlaSer Val Ile His Met Asp Ile Asp Pro Ala Glu 285 290 295 atg aac aag ctgcgt cag gca cat gtg gca tta caa ggt gat tta aat 963 Met Asn Lys Leu ArgGln Ala His Val Ala Leu Gln Gly Asp Leu Asn 300 305 310 315 gct ctg ttacca gca tta cag cag ccg tta aat atc aat gac tgg cag 1011 Ala Leu Leu ProAla Leu Gln Gln Pro Leu Asn Ile Asn Asp Trp Gln 320 325 330 cta cac tgcgcg cag ctg cgt gat gaa cat gcc tgg cgt tac gac cat 1059 Leu His Cys AlaGln Leu Arg Asp Glu His Ala Trp Arg Tyr Asp His 335 340 345 ccc ggt gacgct atc tac gcg cca ttg ttg tta aaa caa ctg tcg gat 1107 Pro Gly Asp AlaIle Tyr Ala Pro Leu Leu Leu Lys Gln Leu Ser Asp 350 355 360 cgt aaa cctgcg gat tgc gtc gtg acc aca gat gtg ggg cag cac cag 1155 Arg Lys Pro AlaAsp Cys Val Val Thr Thr Asp Val Gly Gln His Gln 365 370 375 atg tgg gccgcg cag cac atc gca cac act cgc ccg gaa aat ttc att 1203 Met Trp Ala AlaGln His Ile Ala His Thr Arg Pro Glu Asn Phe Ile 380 385 390 395 acc tccagc ggc tta ggc acc atg ggt ttc ggt tta cca gcg gcg gtt 1251 Thr Ser SerGly Leu Gly Thr Met Gly Phe Gly Leu Pro Ala Ala Val 400 405 410 ggc gcacaa gtc gca cga ccg aac gat act gtc gtc tgt atc tcc ggt 1299 Gly Ala GlnVal Ala Arg Pro Asn Asp Thr Val Val Cys Ile Ser Gly 415 420 425 gac ggctct ttc atg atg aat gtg caa gag ctg ggc acc gta aaa cgc 1347 Asp Gly SerPhe Met Met Asn Val Gln Glu Leu Gly Thr Val Lys Arg 430 435 440 aag cagtta ccg ttg aaa atc gtc tta ctc gat aac caa cgg tta ggg 1395 Lys Gln LeuPro Leu Lys Ile Val Leu Leu Asp Asn Gln Arg Leu Gly 445 450 455 atg gttcga caa tgg cag caa ctg ttt ttt cag gaa cga tac agc gaa 1443 Met Val ArgGln Trp Gln Gln Leu Phe Phe Gln Glu Arg Tyr Ser Glu 460 465 470 475 accacc ctt act gat aac ccc gat ttc ctc atg tta gcc agc gcc ttc 1491 Thr ThrLeu Thr Asp Asn Pro Asp Phe Leu Met Leu Ala Ser Ala Phe 480 485 490 ggcatc cct ggc caa cac atc acc cgt aaa gac cag gtt gaa gcg gca 1539 Gly IlePro Gly Gln His Ile Thr Arg Lys Asp Gln Val Glu Ala Ala 495 500 505 ctcgac acc atg ctg aac agt gat ggg cca tac ctg ctt cat gtc tca 1587 Leu AspThr Met Leu Asn Ser Asp Gly Pro Tyr Leu Leu His Val Ser 510 515 520 atcgac gaa ctt gag aac gtc tgg ccg ctg gtg ccg cct ggc gcc agt 1635 Ile AspGlu Leu Glu Asn Val Trp Pro Leu Val Pro Pro Gly Ala Ser 525 530 535 aattca gaa atg ttg gag aaa tta tca tga tgcaacatca ggtcaatgta 1685 Asn SerGlu Met Leu Glu Lys Leu Ser 540 545 tcggctcgct tcaatccgga aaccttagaacgtgttttac gcgtggtgcg tcatcgtggt 1745 ttccacgtct gctcaatgaa tatggctgccgccagcgatg cacaaaatat aaatatcgaa 1805 ttgaccgttg ccagcccacg gtcggtcgacttactgttta gtcagttaaa taaactggtg 1865 gacgtcgcac acgttgccat ctgccagagcacaaccacat cacaacaaat ccgcgcctga 1925 gcgcaaaagg aatataaaaa tgaccacgaagaaagctgat tacatttggt tcaatgggga 1985 gatggttcgc tgggaagacg cgaaggtgcatgtgatgtcg cacgcgctgc actatggcac 2045 ctcggttttt gaaggcatcc gttgctacgactcacacaaa ggaccggttg tattccgcca 2105 tcgtga 2111 12 548 PRT Escherichiacoli 12 Met Asn Gly Ala Gln Trp Val Val His Ala Leu Arg Ala Gln Gly Val1 5 10 15 Asn Thr Val Phe Gly Tyr Pro Gly Gly Ala Ile Met Pro Val TyrAsp 20 25 30 Ala Leu Tyr Asp Gly Gly Val Glu His Leu Leu Cys Arg His GluGln 35 40 45 Gly Ala Ala Met Ala Ala Ile Gly Tyr Ala Arg Ala Thr Gly LysThr 50 55 60 Gly Val Cys Ile Ala Thr Ser Gly Pro Gly Ala Thr Asn Leu IleThr 65 70 75 80 Gly Leu Ala Asp Ala Leu Leu Asp Ser Ile Pro Val Val AlaIle Thr 85 90 95 Gly Gln Val Ser Ala Pro Phe Ile Gly Thr Asp Ala Phe GlnGlu Val 100 105 110 Asp Val Leu Gly Leu Ser Leu Ala Cys Thr Lys His SerPhe Leu Val 115 120 125 Gln Ser Leu Glu Glu Leu Pro Arg Ile Met Ala GluAla Phe Asp Val 130 135 140 Ala Ser Ser Gly Arg Pro Gly Pro Val Leu ValAsp Ile Pro Lys Asp 145 150 155 160 Ile Gln Leu Ala Ser Gly Asp Leu GluPro Trp Phe Thr Thr Val Glu 165 170 175 Asn Glu Val Thr Phe Pro His AlaGlu Val Glu Gln Ala Arg Gln Met 180 185 190 Leu Ala Lys Ala Gln Lys ProMet Leu Tyr Val Gly Gly Gly Val Gly 195 200 205 Met Ala Gln Ala Val ProAla Leu Arg Glu Phe Leu Ala Thr Thr Lys 210 215 220 Met Pro Ala Thr CysThr Leu Lys Gly Leu Gly Ala Val Glu Ala Asp 225 230 235 240 Tyr Pro TyrTyr Leu Gly Met Leu Gly Met His Gly Thr Lys Ala Ala 245 250 255 Asn PheAla Val Gln Glu Cys Asp Leu Leu Ile Ala Val Gly Ala Arg 260 265 270 PheAsp Asp Arg Val Thr Gly Lys Leu Asn Thr Phe Ala Pro His Ala 275 280 285Ser Val Ile His Met Asp Ile Asp Pro Ala Glu Met Asn Lys Leu Arg 290 295300 Gln Ala His Val Ala Leu Gln Gly Asp Leu Asn Ala Leu Leu Pro Ala 305310 315 320 Leu Gln Gln Pro Leu Asn Ile Asn Asp Trp Gln Leu His Cys AlaGln 325 330 335 Leu Arg Asp Glu His Ala Trp Arg Tyr Asp His Pro Gly AspAla Ile 340 345 350 Tyr Ala Pro Leu Leu Leu Lys Gln Leu Ser Asp Arg LysPro Ala Asp 355 360 365 Cys Val Val Thr Thr Asp Val Gly Gln His Gln MetTrp Ala Ala Gln 370 375 380 His Ile Ala His Thr Arg Pro Glu Asn Phe IleThr Ser Ser Gly Leu 385 390 395 400 Gly Thr Met Gly Phe Gly Leu Pro AlaAla Val Gly Ala Gln Val Ala 405 410 415 Arg Pro Asn Asp Thr Val Val CysIle Ser Gly Asp Gly Ser Phe Met 420 425 430 Met Asn Val Gln Glu Leu GlyThr Val Lys Arg Lys Gln Leu Pro Leu 435 440 445 Lys Ile Val Leu Leu AspAsn Gln Arg Leu Gly Met Val Arg Gln Trp 450 455 460 Gln Gln Leu Phe PheGln Glu Arg Tyr Ser Glu Thr Thr Leu Thr Asp 465 470 475 480 Asn Pro AspPhe Leu Met Leu Ala Ser Ala Phe Gly Ile Pro Gly Gln 485 490 495 His IleThr Arg Lys Asp Gln Val Glu Ala Ala Leu Asp Thr Met Leu 500 505 510 AsnSer Asp Gly Pro Tyr Leu Leu His Val Ser Ile Asp Glu Leu Glu 515 520 525Asn Val Trp Pro Leu Val Pro Pro Gly Ala Ser Asn Ser Glu Met Leu 530 535540 Glu Lys Leu Ser 545 13 2111 DNA Escherichia coli RBS (8)..(12) 13caggacgagg aactaactat gaatggcgca cagtgggtgg tacatgcgtt gcgggcacag 60ggtgtgaaca ccgttttcgg ttatccgggt ggcgcaatta tgccggttta cgatgcattg 120tatgacggcg gcgtggagca cttgctgtgc cgacatgagc agggtgcggc aatggcggct 180atcggttatg cccgtgctac cggcaaaact ggcgtatgta tcgccacgtc tggtccgggc 240gcaaccaacc tgataaccgg gcttgcggac gcactgttag attctatccc tgttgttgcc 300atcaccggtc aagtgtccgc accgtttatc ggcacggacg catttcagga agtggatgtc 360ctgggattgt cgttagcctg taccaagcac agctttctgg tgcagtcgct ggaagagttg 420ccgcgcatta tggctgaagc attcgacgtt gccagctcag gtcgtcctgg tccggttctg 480gtcgatatcc caaaagatat ccagctagcc agcggtgacc tggaaccgtg gttcaccacc 540gttgaaaacg aagtgacttt cccacatgcc gaagttgagc aagcgcgcca gatgctggca 600aaagcgcaaa aaccgatgct gtacgttggt ggtggcgtgg gtatggcgca ggcagttcct 660gctttacgag aatttctcgc taccacaaaa atgcctgcca cctgcacgct gaaagggctg 720ggcgcagttg aagcagatta tccgtactat ctgggcatgc tgggaatgca tggcaccaaa 780gcggcgaact tcgcggtgca ggagtgcgac ttgctgatcg ccgtgggtgc acgttttgat 840gaccgggtga ccggcaaact gaacaccttc gcaccacacg ccagtgttat ccatatggat 900atcgacccgg cagaaatgaa caagctgcgt caggcacatg tggcattaca aggtgattta 960aatgctctgt taccagcatt acagcagccg ttaaatatca atgactggca gctacactgc 1020gcgcagctgc gtgatgaaca tgcctggcgt tacgaccatc ccggtgacgc tatctacgcg 1080ccattgttgt taaaacaact gtcggatcgt aaacctgcgg attgcgtcgt gaccacagat 1140gtggggcagc accagatgtg ggccgcgcag cacatcgcac acactcgccc ggaaaatttc 1200attacctcca gcggcttagg caccatgggt ttcggtttac cagcggcggt tggcgcacaa 1260gtcgcacgac cgaacgatac tgtcgtctgt atctccggtg acggctcttt catgatgaat 1320gtgcaagagc tgggcaccgt aaaacgcaag cagttaccgt tgaaaatcgt cttactcgat 1380aaccaacggt tagggatggt tcgacaatgg cagcaactgt tttttcagga acgatacagc 1440gaaaccaccc ttactgataa ccccgatttc ctcatgttag ccagcgcctt cggcatccct 1500ggccaacaca tcacccgtaa agaccaggtt gaagcggcac tcgacaccat gctgaacagt 1560gatgggccat acctgcttca tgtctcaatc gacgaacttg agaacgtctg gccgctggtg 1620ccgcctggcg ccagtaattc agaaatgttg gagaaattat c atg atg caa cat cag 1676Met Met Gln His Gln 1 5 gtc aat gta tcg gct cgc ttc aat ccg gaa acc ttagaa cgt gtt tta 1724 Val Asn Val Ser Ala Arg Phe Asn Pro Glu Thr Leu GluArg Val Leu 10 15 20 cgc gtg gtg cgt cat cgt ggt ttc cac gtc tgc tca atgaat atg gct 1772 Arg Val Val Arg His Arg Gly Phe His Val Cys Ser Met AsnMet Ala 25 30 35 gcc gcc agc gat gca caa aat ata aat atc gaa ttg acc gttgcc agc 1820 Ala Ala Ser Asp Ala Gln Asn Ile Asn Ile Glu Leu Thr Val AlaSer 40 45 50 cca cgg tcg gtc gac tta ctg ttt agt cag tta aat aaa ctg gtggac 1868 Pro Arg Ser Val Asp Leu Leu Phe Ser Gln Leu Asn Lys Leu Val Asp55 60 65 gtc gca cac gtt gcc atc tgc cag agc aca acc aca tca caa caa atc1916 Val Ala His Val Ala Ile Cys Gln Ser Thr Thr Thr Ser Gln Gln Ile 7075 80 85 cgc gcc tga gcgcaaaagg aatataaaaa tgaccacgaa gaaagctgat 1965Arg Ala tacatttggt tcaatgggga gatggttcgc tgggaagacg cgaaggtgcatgtgatgtcg 2025 cacgcgctgc actatggcac ctcggttttt gaaggcatcc gttgctacgactcacacaaa 2085 ggaccggttg tattccgcca tcgtga 2111 14 87 PRT Escherichiacoli 14 Met Met Gln His Gln Val Asn Val Ser Ala Arg Phe Asn Pro Glu Thr1 5 10 15 Leu Glu Arg Val Leu Arg Val Val Arg His Arg Gly Phe His ValCys 20 25 30 Ser Met Asn Met Ala Ala Ala Ser Asp Ala Gln Asn Ile Asn IleGlu 35 40 45 Leu Thr Val Ala Ser Pro Arg Ser Val Asp Leu Leu Phe Ser GlnLeu 50 55 60 Asn Lys Leu Val Asp Val Ala His Val Ala Ile Cys Gln Ser ThrThr 65 70 75 80 Thr Ser Gln Gln Ile Arg Ala 85

What is claimed is:
 1. Process for the preparation of D-pantothenic acid and/or alkaline earth metal salts thereof or feedstuffs additives comprising these by fermentation of microorganisms of the Enterobacteriaceae family, in particular those which already produce D-pantothenic acid, wherein a) at least the nucleotide sequence(s) in the microorganisms which code(s) for the pckA gene is (are) attenuated, in particular eliminated, b) the D-pantothenic acid and/or salts thereof is (are) concentrated in the medium or in the cells of the microorganisms, and c) after conclusion of the fermentation, the desired products are isolated, the biomass and/or further constituents of the fermentation broth being left in the product or optionally being separated off completely or in part.
 2. Process according to claim 1, wherein the fermentation is carried out in the presence of alkaline earth metal salts, these being added continuously or discontinuously in stoichiometric amounts in particular, and a product comprising alkaline earth metal salts of D-pantothenic acid or consisting of these being obtained.
 3. Process according to claim 1, wherein the microorganisms of the Enterobacteriaceae family belong to the genus Escherichia.
 4. Process according to claim 3, wherein the microorganisms originate from the genus Escherichia, in particular the species Escherichia coli.
 5. Process according to claim 1, wherein in addition to attenuation of the pckA gene, one or more genes, chosen from the group, is (are) enhanced, in particular over-expressed: 5.1 the ilvGM operon which codes for acetohydroxy-acid synthase II, 5.2 the panB gene which codes for ketopantoate hydroxymethyl transferase, 5.3 the panE gene which codes for ketopantoate reductase, 5.4 the panD gene which codes for aspartate decarboxylase, 5.5 the panC gene which codes for pantothenate synthetase, 5.6 the serC gene which codes for phosphoserine transaminase, 5.7 the gcvT, gcvH and gcvP genes which code for the glycine cleavage system, individually or together, 5.8 the glyA gene which codes for serine hydroxymethyl transferase.
 6. Process according to claim 1, wherein bacteria are employed in which the metabolic pathways which reduce the formation of D-pantothenic acid are at least partly eliminated, in particular the 6.1 avtA gene which codes for transaminase C and 6.2 the poxB gene which codes for pyruvate oxidase.
 7. Process according to claim 1, wherein the expression of the polynucleotide (s) which code(s) for the pckA gene is attenuated, in particular eliminated.
 8. Process for the preparation of feedstuffs additives comprising D-pantothenic acid and/or salts thereof, according to claim 1, wherein a) optionally all or some of the biomass and/or a portion of the constituents are separated off from a D-pantothenic acid-containing fermentation broth obtained by fermentation, b) the mixture obtained in this way is optionally concentrated, and c) the feedstuffs additive comprising the pantothenic acid and/or the pantothenate is converted into a free-flowing form by suitable measures, and d) a free-flowing animal feedstuffs additive with a particle size distribution of 20 to 2000 μm is obtained by suitable measures.
 9. Process for the preparation of animal feedstuffs additives according to claim 8 with a content of D-pantothenic acid and/or salts thereof, chosen from the group consisting of the magnesium or calcium salt, in the range from about 20 to 80 wt. % (dry mass) from fermentation broths, comprising the steps of a) optionally removal of water from the fermentation broth (concentration). b) removal of an amount of ≧0 to 100% of the biomass formed during the fermentation, c) optionally addition of one or more of the compounds mentioned to the fermentation broths obtained according to a) and b), the amount of compounds added being such that the total concentration thereof in the animal feedstuffs additive is in the range from about 20 to 80 wt. %, and d) obtaining of the animal feedstuffs additive in the desired powder or, preferably, granule form.
 10. Process according to claim 8, wherein an animal feedstuffs additive with the desired particle size is obtained from the fermentation broth, optionally after addition of D-pantothenic acid and/or salts thereof and optionally after addition of organic and inorganic auxiliary substances, by a) drying and compacting, or b) spray drying, or c) spray drying and granulation, or d) spray drying and build-up granulation.
 11. Microorganisms of the Enterobacteriaceae family which produce pantothenic acid and in which the pckA gene is present in attenuated form.
 12. Microorganisms of the Enterobacteriaceae family which produce pantothenic acid and in which the pckA gene is present in eliminated form.
 13. Microorganisms of the Enterobacteriaceae family which produce pantothenic acid, according to claim 12, wherein the pckA gene contains a deletion according to SEQ ID No.
 8. 